Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
九州大学学術情報リポジトリKyushu University Institutional Repository
Senescence and odontoblastic differentiation ofdental pulp cells
Nozu, AoiDepartment of Endodontology and Operative Dentistry, Faculty of Dental Science, KyushuUniversity
Hamano, SayuriDepartment of Endodontology and Operative Dentistry, Faculty of Dental Science, KyushuUniversity
Tomokiyo, AtsushiDivision of Endodontology, Kyushu University Hospital
Hasegawa, DaigakuDivision of Endodontology, Kyushu University Hospital
他
http://hdl.handle.net/2324/2198478
出版情報:Journal of cellular physiology. 234 (1), pp.849-859, 2018-08-04. Wistar Institute ofAnatomy and Biologyバージョン:権利関係:
Senescence and odontoblastic differentiation of dental pulp
cells
Aoi Nozu1, Sayuri Hamano1,2, Atsushi Tomokiyo3, Daigaku Hasegawa3, Hideki Sugii1,
Shinichiro Yoshida1, Hiromi Mitarai1, Shuntaro Taniguchi1, Naohisa Wada4, Hidefumi
Maeda1,3*
1Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka,
Japan 2OBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan 3Division of Endodontology, Kyushu University Hospital, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka, Japan 4Division of General Dentistry, Kyushu University Hospital, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka, Japan
*Corresponding author: Hidefumi Maeda, DDS, PhD
Department of Endodontology and Operative Dentistry
Faculty of Dental Science, Kyushu University
3-1-1 Maidashi Fukuoka 812-8582, Japan.
Phone: +81-92-642-6432
Fax: +81-92-642-6366
E-mail: [email protected]
Total Number of Table/Figures: 1 table and 13 figures (including 7 supplementary
figures)
Keywords: Dental pulp cell; Odontoblast; Senescence; Tumor Necrosis Factor-alpha;
Tumor Necrosis Factor Receptor 1
A running head: differentiation of dental pulp cells
Abstract
Cellular senescence has been suggested to be involved in physiological changes of
cytokine production. Previous studies showed that the concentration of tumor necrosis
factor- (TNF-) is higher in the blood of aged people compared with that of young
people. So far, the precise effects of TNF- on odontoblastic differentiation of pulp cells
has been controversial. Therefore, we aimed to clarify how this cytokine affected pulp
cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the
plateau of their growth, and the cells were isolated at actively (yHDPCs) or inactively
(sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while
yHDPCs did not. When these HDPCs were cultured in odontoblast-inductive medium,
both young and senescent cells showed mineralization, but mineralization in sHDPCs was
lower compared with yHDPCs. However, the administration of TNF- to this culture
medium altered these responses: yHDPCs showed downregulated mineralization, while
sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of
TNFR1, a receptor of TNF-, was significantly upregulated in sHDPCs compared with
yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-
-treated sHDPCs, whereas restored the reduction in TNF--treated yHDPCs. These
results suggested that sHDPCs preserved the odontoblastic differentiation capacity, and
TNF- promoted odontoblastic differentiation of HDPCs with progress of their
population doublings through increased expression of TNFR1. Thus, TNF- might exert
a different effect on odontoblastic differentiation of HDPCs depending on their
proliferating activity. In addition, the calcification of pulp chamber with age may be
related with increased reactivity of pulp cells to TNF-.
Introduction
Dental pulp cells have a high repair ability but do not mineralize under physiological
conditions. However, under pathologic conditions, the protective response, such as
reparative dentin formation, is induced by inflammatory stimuli during pulp wound
healing (Goldberg et al., 2008) or mechanical stimuli. In this protective event,
odontoblast-like cells are differentiated from the progenitor cells in the pulp tissue and
secrete dentin matrices (Dimitrova-Nakov et al., 2014). These matrices include
extracellular matrix proteins, such as bone sialoprotein (BSP), dentin sialophosphoprotein
(DSPP), and osteocalcin (OCN), which are involved in odontoblastic differentiation
(Chen et al., 1992; Suzuki et al., 2012; Wei et al., 2007). In addition, the expression of
Nestin, which forms type VI V Iintermediate filament proteins, is also related to
odontoblastic differentiation (Lee et al., 2012).
The protective response of dental pulp is mediated by inflammatory molecules such as
tumor necrosis factor- (TNF-) derived from immune cells (Jontell et al., 1998). Several
studies have examined the involvement of TNF- in odontoblastic differentiation. One
study reported that TNF- suppressed mineralization in an odontoblast-like cell line
(Nakayama et al., 2016), whereas another study showed that TNF- promoted osteogenic
differentiation of human dental pulp cells (Paula-Silva et al., 2009). Thus, whether TNF-
can induce odontoblastic differentiation of dental pulp cells remains unclear.
Tumor suppressors, such as p16, p21 and p53, increase in gene expression with age and
are known senescence-related genes (Bringold and Serrano, 2000; Johmura et al., 2014).
Cellular senescence is also characterized by an accumulation of senescence-associated β-
galactosidase (SA--gal) activity (Dimri et al., 1995). Senescent cells secrete higher
levels of proteins with a senescence-associated secretory phenotype, such as IL-6, TNF-
, MMPs, monocyte chemoattractant protein-1, and IGF binding proteins, in multiple
tissues with age (Coppe et al., 2008). A relationship between aging and chronic
inflammation has been demonstrated (Franceschi et al., 2000), and the concentration of
the TNF- inflammatory molecule was shown to be increased in blood in older people
compared with that in the young (Paolisso et al., 1998b). However, whether dental pulp
cells show a different response to TNF- depending on senescence remains unknown.
In this study, we hypothesized that odontoblastic differentiation of dental pulp cells
might be promoted by TNF- depending on their senescent stage. Therefore, the aim of
this study was to investigate how the population doubling of dental pulp cells affected
their reactivity against TNF- on their odontoblastic differentiation and to examine the
possible underlying mechanism.
Materials and methods
Cell culture
Three lines of human dental pulp cells (HDPCs) were isolated from healthy human
premolars of a 24-year-old male (HDPC-3R), a 21-year-old female (HDPC-3Q), and a
23-year-old male (HDPC-3S) who visited Kyushu University hospital for tooth extraction,
with informed consent. HDPCs were cultured in -minimal essential medium (-MEM;
Gibco-BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS; Biowest,
Nuaille, France) at 37C in a humidified atmosphere of 5% CO2 and 95% air. All
procedures were performed in compliance with the Research Ethics Committee, Kyushu
University (No.20A-3).
Determination of population doublings
Population doublings (PDs) of HDPCs were determined until cells reached a plateau in
proliferation. PD was calculated by the following formula: [log10Nh-log10Ns] /
log10[2], in which Nh and Ns indicate the cell number at harvest and at seeding,
respectively. PD was calculated by adding the PD of the previous passage to the PD
calculated at each passage.
Senescence assay
SA--gal activity was measured by the β-Galactosidase Staining Kit (Cell Signaling
Technology, Inc., Danvers, MA) following the manufacturer’s protocol. Briefly, cells
were fixed in 2% formaldehyde and 0.2% glutaraldehyde. After cells were washed, they
were incubated overnight in the staining solution (1 mg/mL 5-bromo-4-chloro-3-indolyl
h-D-galactosidase, 40 mM citric acid, pH 6.0, 40 mM sodium phosphate, pH 6.0, 5 mM
potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2
mM magnesium chloride) at 37°C. Under a microscope (Keyence, Osaka, Japan), the
senescent cells appeared blue.
Odontoblastic differentiation of HDPCs
HDPCs were cultured using three different medium conditions: 10% FBS/-MEM as
control culture medium (CM), CM containing 2 mM CaCl2 as differentiation medium
(DM), or DM containing TNF- (DM+TNF-). Our recent studies showed that DM
promoted odontoblastic differentiation of HDPCs (Mizumachi et al., 2017; Serita et al.,
2017). After culturing, the cells were subjected to Alizarin red S staining and PCR
analysis.
Alizarin red S staining
HDPCs were fixed for 1 h with 10% formalin. After washing with sterile water, the cells
were incubated with Alizarin red S solution (pH 4.1–4.3) (Sigma-Aldrich, Tokyo, Japan)
for 1 h. Each Alizarin red-positive region was evaluated using a microscope (Keyence).
Quantitative RT-PCR
Total cellular RNA was harvested using TRIzol Reagent (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1
mg total RNA using an ExScript RT Reagent kit (Takara Bio Inc., Shiga, Japan).
Quantitative RT-PCR was performed with a SYBR Green II RT-PCR kit (Takara Bio Inc.)
using a Thermal Cycler Dice Real Time System (Takara Bio Inc.) under the following
conditions: 95 C for 10 s and then 40 cycles at 95 C for 5 s and 60 C for 30 s, followed
by a dissociation program at 95 C for 15 s, 60 C for 30 s and 60 C for 30 s and 95 C
for 15 s. Specific primer sequences, annealing temperatures, and product sizes for each
gene are listed in a TABLE. -actin served as an internal control. Expression levels of the
target genes were calculated using Ct values.
siRNA transfection
HDPCs were transfected with TNFR1 siRNA (MISSION siRNA; Hs_TNFRSF1A_3459;
Sigma) or control siRNA (MISSION siRNA Universal Negative Control 1; SIC-001-10;
Sigma) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s
instructions. Briefly, cells at 80% confluence were cultured in Opti-MEM I (Invitrogen)
containing 10% FBS. A siRNA–lipid complex, comprising 10 pmol siRNA and 3 µl of
Lipofectamine RNAiMAX in 50 µl of Opti-MEM, was prepared. After incubation for 5
min at room temperature, the complex was added to the cells and cells were incubated for
24 h.
Statistical analysis
All data are presented as the mean SD. Statistical analyses were performed by one-way
ANOVA followed by Tukey’s test for multiple comparisons. Student’s unpaired t-test was
performed for comparisons of two mean values. P < 0.05 was considered statistically
significant.
Results
Determination of PDs of HDPCs and the expression of aging-related markers
PDs of HDPCs cultured in 10% FBS/-MEM were determined by culturing until the cell
proliferation reached a plateau (Fig. 1A and Supplementary Fig. 1A, E). In HDPC-3R
cells, the cell proliferation activity after PD25 was diminished (Fig. 1A). The cells
isolated at PD6 showed active proliferation, while the cells at PD28 had almost reached
a plateau of growth (Fig. 1A). To verify if the cells at PD28 were senescent, SA-β-gal
activity was examined and the results demonstrated that PD28 cells showed positive SA-
β-gal activity whereas PD6 cells did not (Fig. 1B). In addition, RT-PCR analysis of aging-
related markers, p16, p21, and p53 showed that the expression levels of these genes in
HDPC-3R cells at PD28 were significantly upregulated compared with the cells at PD6
(Fig. 2C). The other two HDPC lines, HDPCs-3Q and -3S, also showed the same results;
HDPC-3Q at PD6 and HDPC-3S at PD5 showed active proliferation while HDPC-3Q at
PD27 and HDPC-3S at PD26 exhibited downregulated proliferation and upregulated
senescence markers (Supplementary Fig. 1A–F). Based on these data, HDPCs at actively
and inactively proliferating stages were used as young HDPCs (yHDPCs) and senescent
HDPCs (sHDPCs), respectively, in the following analyses.
Expression of odontoblast-related genes in yHDPCs and sHDPCs
The expression of odontoblast-related genes, such as BSP, DSPP, Nestin, and OCN was
investigated in yHDPCs and sHDPCs cultured in 10 FBS/-MEM (Fig. 2 and
Supplementary Fig. 2). The results showed that the expression levels of these genes were
significantly higher in sHDPCs than in yHDPCs.
Effects of TNF- on odontoblastic differentiation of HDPCs
We recently showed that DM promoted odontoblastic differentiation of HDPCs
(Mizumachi et al., 2017; Serita et al., 2017). To examine the effects of TNF- on
odontoblastic differentiation of yHDPCs and sHDPCs, the cells were cultured in CM or
DM with or without TNF- for 7 days. Both yHDPCs and sHDPCs cultured in CM alone
formed few alizarin red S-positive mineralized deposits, while both cells cultured in DM
alone showed increased mineralized deposits (Fig. 3A, D and Supplementary Fig. 3A, D,
G, J). However, sHDPCs treated with TNF- revealed enhanced mineralization upon
differentiation with DM, whereas TNF--treated yHDPCs showed reduced
mineralization compared with the cultures in DM (Fig. 3A, B, D, E and Supplementary
Fig. 3A, B, D, E, G, H, J, K). Furthermore, the expression of odontoblast-related genes,
such as BSP, DSPP, Nestin, and OCN, in both yHDPCs and sHDPCs cultured under the
same conditions also showed similar results as the mineralization data (Fig. 3C, F and
Supplementary Fig. 3C, F, I, L).
Comparison of the mineralization ratio between yHDPCs and sHDPCs
The mineralization area in yHDPCs and sHDPCs cultured in CM or DM with or without
TNF- was first determined and then the cell number in each condition was counted.
From these data, the mineralization ratio per cell was calculated. The results showed that
sHDPCs demonstrated significantly higher mineralization activity per cell under TNF-
treatment compared with yHDPCs (Fig. 4 and Supplementary Fig. 4A, B).
Contribution of TNFR1 to TNF--induced odontoblastic features in sHDPCs.
Gene expression of a TNF- receptor, TNFR1, was examined in yHDPCs and sHDPCs
(Fig. 5A and Supplementary Fig. 5A, F). sHDPCs were found to express TNFR1 at a
higher level compared with yHDPCs (Fig. 5A and Supplementary Fig. 5A, F). To further
examine whether the effect of TNF- on the odontoblastic differentiation of sHDPCs was
through the TNFR1 receptor, the cells were transduced with TNFR1 siRNA or control
siRNA for 24 h. The suppression of TNFR1 in the transduced sHDPCs was first confirmed
by quantitative RT-PCR assays (Fig. 5B and Supplementary Fig. 5B, G). Then, the
transduced sHDPCs were cultured in CM or DM with or without TNF- for 9 days.
sHDPCs cultured in DM showed increased alizarin red S-positive deposits, whereas the
cells cultured in CM showed no positive reaction (Fig. 5C, D and Supplementary Fig. 5C,
D, H, I). Control and TNFR1 siRNA-transduced sHDPCs in DM without TNF- showed
similar mineralization levels (Fig. 5C, D and Supplementary Fig. 5C, D, H, I). However,
when cultured in DM with TNF-, siTNFR1-transduced sHDPCs showed significantly
reduced mineralization compared with control siRNA-transduced cells (Fig. 5C, D and
Supplementary Fig. 5C, D, H, I). The expression of odontoblast-related genes, such as
BSP, DSPP, Nestin, and OCN, in sHDPCs cultured under the same conditions also showed
similar results (Fig. 5E and Supplementary Fig. 5E, J).
Contribution of TNFR1 to TNF--reduced odontoblastic features in yHDPCs.
The effect of TNF- on the odontoblastic differentiation of yHDPCs through the TNFR1
was also investigated by the same methods using the transduction with TNFR1 siRNA or
control siRNA as described above. Downregulation of TNFR1 in the transduced yHDPCs
was verified by quantitative RT-PCR assays (Fig. 6A and Supplementary Fig. 6A, E).
Although yHDPCs cultured in CM showed no alizarin red S-positive reaction, the cells
cultured in DM showed increased mineralization (Fig. 6B, C and Supplementary Fig 6B,
C, F, G). Control and TNFR1 siRNA-transduced yHDPCs showed similar degree of
mineralization levels in DM without TNF- (Fig. 6B, C and Supplementary Fig 6B, C, F,
G). However, when cultured in DM with TNF-, control siRNA-transduced yHDPCs
reduced mineralization, whereas the transduction of siTNFR1 restored it (Fig. 6B, C and
Supplementary Fig 6B, C, F, G). BSP, DSPP, Nestin, and OCN, in yHDPCs cultured under
the same conditions also exhibited corresponding results (Fig. 6D and Supplementary Fig.
6D, H).
Discussion
In the present study, we first clarified that HDPCs showed odontoblastic features with
increasing PDs, and furthermore that the reactivity of these cells against TNF- in terms
of odontoblastic differentiation with TNF- was promoted through increased expression
of TNFR1 and its altered signaling pathway, depending on their PD. Our results indicated
that HDPCs form the calcified bodies such as denticles and pulp stones in the pulp
chamber with age.
A recent study demonstrated that normal human diploid fibroblasts exposed to various
senescence-inducing stimuli showed the same senescent features as nevus cells that were
oncogene-induced senescent cells in vivo (Johmura et al., 2014). Thus, to obtain sHDPCs,
we performed a long-term culture and found that HDPCs whose proliferation ratio was
decreased accumulated SA--gal activity and gene expression of aging-related markers.
A previous report showed that dental pulp cells of aged rats showed increased
mineralization and ALP activity compared with those of young rats. (Ma et al., 2009).
However, the mechanism was unclear. Our present study revealed that sHDPCs showed
enhanced expression of odontoblast-related genes compared with yHDPCs, suggesting
that HDPCs might acquire odontoblastic features with aging.
Several studies demonstrated that the TNF- concentration in human plasma was
positively associated with advancing age (Michaud et al., 2013; Paolisso et al., 1998a).
Furthermore, a recent study demonstrated that TNF- promoted the senescence of
nucleus pulposus cells (Li et al., 2017). Interestingly, we found that the gene expression
of TNF- in sHDPCs was upregulated compared with that in yHDPCs, indicating the
association of TNF- with aging of HDPCs (Supplementary Fig. 7).
Several studies had previously investigated the roles of TNF- in the odontoblastic
differentiation of dental pulp cells, however the results were controversial. For example,
one study showed that TNF- suppressed mineralization in an odontoblast-like cell line
(Nakayama et al., 2016), whereas another report stated that TNF- promoted osteogenic
differentiation of HDPCs (Paula-Silva et al., 2009). Although the discrepancy between
these studies might depend on the PD and differentiation stages of the cells, the
mechanism remained unclear. In our study, sHDPCs treated with TNF- further promoted
odontoblastic differentiation whereas TNF- treatment reduced it in yHDPCs. A recent
study revealed that young and old dental pulp cells under low-grade inflammation present
similar levels of ALP activity, whereas in irreversible pulpitis, the old pulp cells showed
significantly upregulated ALP activity compared with young cells (Aslantas et al., 2016).
These data could argue the possible involvement of different signaling pathways in TNF-
-inducing events depending on the cell aging stage, which might cause different cell
responses.
We further focused on what contributed to the increased reactivity of sHDPCs against
TNF-, compared with yHDPCs. Our present results demonstrated that HDPCs showed
increased TNFR1 expression with increasing PDs and that enhanced TNFR1 expression
was involved in TNF--induced odontoblastic differentiation of sHDPCs. These findings
suggested that the increased expression of TNFR1 may play pivotal roles in promoting
odontoblastic differentiation of dental pulp cells of aged people. On the other hand,
although the expression of TNFR1 in yHDPCs was lower than that of sHDPCs, TNFR1
was also involved in TNF--reduced odontoblastic differentiation in yHDPCS. This
paradoxical result suggested the different signaling pathways of TNF- via TNFR1
between yHDPCs and sHDPCs. A previous study reported that human fibroblasts from
aged individuals showed increased levels of NF-B activation and increased
inflammatory gene expression when compared with cells derived from young individuals
(Adler et al., 2007). In addition, NF-B/p65 DNA binding increased with age in several
tissues including skin, liver, kidney, cardiac muscle, and gastric mucosa (Helenius et al.,
1996b;Xiao and Majumdar, 2000; Helenius et al., 1996a). Thus such pathway might
contribute to TNF--treated HDPCs.
In conclusion, this study revealed that sHDPCs showed increased odontoblastic
features and that TNF- promoted odontoblastic differentiation of sHDPCs via the
increased expression of TNFR1 and its altered signaling pathway that were associated
with their senescence.
These results provide further understanding of the detailed mechanism of odontoblastic
differentiation with aging and will provide beneficial information for the development of
various preservative treatments of dental pulp tissue. Further studies are needed to clarify
the age-dependent signaling pathways of dental pulp cells.
Acknowledgments
The authors acknowledge Drs. H. Mizumachi, S. Serita, M. Arima, M. Ogawa, S. Fujino,
T. Itoyama, T. Ono and K. Ipposhi for their support. This study was financially supported
by Grants-in-Aid for Scientific Research (15H05023, 16K20457, 16K20458, 17H01598,
17H04385) from the Japan Society for the Promotion of Science. The authors declare no
potential conflicts of interest with respect to the authorship and/or publication of this
article. We thank Gabrielle White Wolf, PhD, from Edanz Group
(www.edanzediting.com/ac) for editing a draft of this manuscript.
References
Adler AS, Sinha S, Kawahara TL, Zhang JY, Segal E, Chang HY. 2007. Motif module map
reveals enforcement of aging by continual NF-kappaB activity. Genes & development
21(24):3244-3257.
Aslantas EE, Buzoglu HD, Karapinar SP, Cehreli ZC, Muftuoglu S, Atilla P, Aksoy Y. 2016.
Age-related Changes in the Alkaline Phosphatase Activity of Healthy and Inflamed
Human Dental Pulp. Journal of endodontics 42(1):131-134.
Bringold F, Serrano M. 2000. Tumor suppressors and oncogenes in cellular senescence.
Experimental gerontology 35(3):317-329.
Chen J, Shapiro HS, Sodek J. 1992. Development expression of bone sialoprotein mRNA in
rat mineralized connective tissues. Journal of bone and mineral research : the official
journal of the American Society for Bone and Mineral Research 7(8):987-997.
Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez PY, Campisi
J. 2008. Senescence-associated secretory phenotypes reveal cell-nonautonomous
functions of oncogenic RAS and the p53 tumor suppressor. PLoS biology 6(12):2853-
2868.
Dimitrova-Nakov S, Baudry A, Harichane Y, Kellermann O, Goldberg M. 2014. Pulp stem
cells: implication in reparative dentin formation. Journal of endodontics 40(4
Suppl):S13-18.
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj
I, Pereira-Smith O, et al. 1995. A biomarker that identifies senescent human cells in
culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of
the United States of America 92(20):9363-9367.
Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, De Benedictis G.
2000. Inflamm-aging. An evolutionary perspective on immunosenescence. Annals of
the New York Academy of Sciences 908:244-254.
Goldberg M, Farges JC, Lacerda-Pinheiro S, Six N, Jegat N, Decup F, Septier D, Carrouel F,
Durand S, Chaussain-Miller C, Denbesten P, Veis A, Poliard A. 2008. Inflammatory
and immunological aspects of dental pulp repair. Pharmacological research 58(2):137-
147.
Helenius M, Hanninen M, Lehtinen SK, Salminen A. 1996a. Aging-induced up-regulation of
nuclear binding activities of oxidative stress responsive NF-kB transcription factor
in mouse cardiac muscle. Journal of molecular and cellular cardiology 28(3):487-498.
Helenius M, Hanninen M, Lehtinen SK, Salminen A. 1996b. Changes associated with aging
and replicative senescence in the regulation of transcription factor nuclear factor-
kappa B. The Biochemical journal 318 ( Pt 2):603-608.
Hess K, Ushmorov A, Fiedler J, Brenner RE, Wirth T. 2009. TNFalpha promotes osteogenic
differentiation of human mesenchymal stem cells by triggering the NF-kappaB
signaling pathway. Bone 45(2):367-376.
Johmura Y, Shimada M, Misaki T, Naiki-Ito A, Miyoshi H, Motoyama N, Ohtani N, Hara E,
Nakamura M, Morita A, Takahashi S, Nakanishi M. 2014. Necessary and sufficient
role for a mitosis skip in senescence induction. Molecular cell 55(1):73-84.
Jontell M, Okiji T, Dahlgren U, Bergenholtz G. 1998. Immune defense mechanisms of the
dental pulp. Critical reviews in oral biology and medicine : an official publication of
the American Association of Oral Biologists 9(2):179-200.
Lee Y, Go EJ, Jung HS, Kim E, Jung IY, Lee SJ. 2012. Immunohistochemical analysis of
pulpal regeneration by nestin expression in replanted teeth. International endodontic
journal 45(7):652-659.
Li P, Gan Y, Xu Y, Song L, Wang L, Ouyang B, Zhang C, Zhou Q. 2017. The inflammatory
cytokine TNF-alpha promotes the premature senescence of rat nucleus pulposus cells
via the PI3K/Akt signaling pathway. Scientific reports 7:42938.
Ma D, Ma Z, Zhang X, Wang W, Yang Z, Zhang M, Wu G, Lu W, Deng Z, Jin Y. 2009. Effect of
Age and Extrinsic Microenvironment on the Proliferation and Osteogenic
Differentiation of Rat Dental Pulp Stem Cells In Vitro. Journal of endodontics
35(11):1546-1553.
Michaud M, Balardy L, Moulis G, Gaudin C, Peyrot C, Vellas B, Cesari M, Nourhashemi F.
2013. Proinflammatory cytokines, aging, and age-related diseases. Journal of the
American Medical Directors Association 14(12):877-882.
Mizumachi H, Yoshida S, Tomokiyo A, Hasegawa D, Hamano S, Yuda A, Sugii H, Serita S,
Mitarai H, Koori K, Wada N, Maeda H. 2017. Calcium-sensing receptor-ERK
signaling promotes odontoblastic differentiation of human dental pulp cells. Bone
101:191-201.
Nakayama K, Hirata-Tsuchiya S, Okamoto K, Morotomi T, Jimi E, Kitamura C. 2016. The
Novel NF-kappaB Inhibitor, MTI-II Peptide Anti-Inflammatory Drug, Suppresses
Inflammatory Responses in Odontoblast-Like Cells. Journal of cellular biochemistry
117(11):2552-2558.
Paolisso G, Rizzo MR, Mazziotti G, Tagliamonte MR, Gambardella A, Rotondi M, Carella C,
Giugliano D, Varricchio M, D'Onofrio F. 1998a. Advancing age and insulin resistance:
role of plasma tumor necrosis factor-alpha. The American journal of physiology 275(2
Pt 1):E294-299.
Paolisso G, Rizzo MR, Mazziotti G, Tagliamonte MR, Gambardella A, Rotondi M, Carella C,
Giugliano D, Varricchio M, D’Onofrio F. 1998b. Advancing age and insulin resistance:
role of plasma tumor necrosis factor-α. American Journal of Physiology -
Endocrinology And Metabolism 275(2):E294-E299.
Paula-Silva FW, Ghosh A, Silva LA, Kapila YL. 2009. TNF-alpha promotes an odontoblastic
phenotype in dental pulp cells. Journal of dental research 88(4):339-344.
Serita S, Tomokiyo A, Hasegawa D, Hamano S, Sugii H, Yoshida S, Mizumachi H, Mitarai H,
Monnouchi S, Wada N, Maeda H. 2017. Transforming growth factor-beta-induced
gene product-h3 inhibits odontoblastic differentiation of dental pulp cells. Archives
of oral biology 78:135-143.
Suzuki S, Haruyama N, Nishimura F, Kulkarni AB. 2012. Dentin sialophosphoprotein and
dentin matrix protein-1: Two highly phosphorylated proteins in mineralized tissues.
Archives of oral biology 57(9):1165-1175.
Wei X, Ling J, Wu L, Liu L, Xiao Y. 2007. Expression of mineralization markers in dental
pulp cells. Journal of endodontics 33(6):703-708.
Xiao ZQ, Majumdar AP. 2000. Induction of transcriptional activity of AP-1 and NF-kappaB
in the gastric mucosa during aging. American journal of physiology Gastrointestinal
and liver physiology 278(6):G855-865.
Figure legends
Fig. 1. Senescence of cultured human dental pulp cells (HDPCs). (A) HDPCs were
cultured until reaching a plateau in proliferation and their population doublings (PDs)
were determined. HDPCs at PD6 showed active proliferation and HDPCs at PD28 just
reached the plateau in proliferation. (B) SA--gal activity in HDPCs at PD6 and PD28
was examined. Only senescent cells were observed to be blue. Bars: 100 µm. (C)
Quantitative RT-PCR assays of p16, p21, and p53 in HDPCs at PD6 and PD28. Data are
shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 2. Comparison of the expression of odontoblast-related genes in young human dental
pulp cells (HDPCs) and senescent HDPCs. Quantitative RT-PCR results of the expression
of odontoblast-related genes BSP, DSPP, Nestin, and OCN are shown. Data are shown as
mean ± standard deviation (n = 3). **p < 0.01.
Fig. 3. Effects of TNF- on odontoblastic differentiation of human dental pulp cells
(HDPCs.) (A, D) Alizarin red S staining of young HDPCs (yHDPCs) (A) and senescent
HDPCs (sHDPCs) (B) cultured in control culture medium (CM) or differentiation
medium (DM) with or without TNF- (10 ng/ml) for 7 days. (B, E) The graphs show the
quantification of the Alizarin red-S positive areas in yHDPCs (B) and sHDPCs (E). (C,
F) Quantitative RT-PCR was performed to examine the expression of odontoblast-related
genes BSP, DSPP, Nestin, and OCN in yHDPCs (C) and sHDPCs (F) cultured in CM or
DM with or without TNF- (10 ng/ml) for 5 days. Data are shown as mean ± standard
deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 4. Comparison of mineralization ratio between yHDPCs and sHDPCs. Alizarin red
S positive areas in yHDPCs and sHDPCs cultured in control culture medium (CM) or
differentiation medium (DM) with or without TNF- for 7 days were determined and
mineralization ratio per cell was calculated. Data are shown as mean ± standard deviation
(n = 3). **p < 0.01.
Fig. 5. Contribution of TNFR1 to odontoblastic differentiation of sHDPCs treated with
TNF-. (A) Quantitative RT-PCR of TNFR1 in yHDPCs and sHDPCs. (B) sHDPCs were
transduced with TNFR1 siRNA (siTNFR1) or control siRNA (Cont), and suppression of
TNFR1 was confirmed by quantitative RT-PCR assays. (C) Alizarin red S staining of the
transduced sHDPCs cultured in control culture medium (CM) or differentiation medium
(DM) with or without TNF- (10 ng/ml) for 9 days. (D) The graph shows the
quantification of the Alizarin red S positive areas. (E) Quantitative RT-PCR of the
expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN in the transduced
sHDPC-3R in CM or DM with or without TNF- (10 ng/ml) for 5 days. Data are shown
as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 6. Contribution of TNFR1 to odontoblastic differentiation of yHDPCs treated with
TNF-. (A) yHDPCs were transduced with TNFR1 siRNA (siTNFR1) or control siRNA
(Cont), and suppression of TNFR1 was confirmed by quantitative RT-PCR assays. (B)
Alizarin red S staining of the transduced yHDPCs cultured in control culture medium
(CM) or differentiation medium (DM) with or without TNF- (10 ng/ml) for 9 days. (C)
The graph shows the quantification of the Alizarin red S positive areas. (D) Quantitative
RT-PCR of the expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN in
the transduced yHDPC-3R in CM or DM with or without TNF- (10 ng/ml) for 5 days.
Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 1. Senescence of cultured human dental pulp cells (HDPCs). (A) HDPCs were
cultured until reaching a plateau in proliferation and their population doublings (PDs)
were determined. HDPCs at PD6 showed active proliferation and HDPCs at PD28 just
reached the plateau in proliferation. (B) SA--gal activity in HDPCs at PD6 and PD28
was examined. Only senescent cells were observed to be blue. Bars: 100 µm. (C)
Quantitative RT-PCR assays of p16, p21, and p53 in HDPCs at PD6 and PD28. Data are
shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 2. Comparison of the expression of odontoblast-related genes in young human dental
pulp cells (HDPCs) and senescent HDPCs. Quantitative RT-PCR results of the
expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN are shown. Data
are shown as mean ± standard deviation (n = 3). **p < 0.01.
Fig. 3. Effects of TNF- on odontoblastic differentiation of human dental pulp cells
(HDPCs.) (A, D) Alizarin red S staining of young HDPCs (yHDPCs) (A) and senescent
HDPCs (sHDPCs) (B) cultured in control culture medium (CM) or differentiation
medium (DM) with or without TNF- (10 ng/ml) for 7 days. (B, E) The graphs show the
quantification of the Alizarin red-S positive areas in yHDPCs (B) and sHDPCs (E). (C,
F) Quantitative RT-PCR was performed to examine the expression of odontoblast-related
genes BSP, DSPP, Nestin, and OCN in yHDPCs (C) and sHDPCs (F) cultured in CM or
DM with or without TNF- (10 ng/ml) for 5 days. Data are shown as mean ± standard
deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 4. Comparison of mineralization ratio between yHDPCs and sHDPCs. Alizarin red
S positive areas in yHDPCs and sHDPCs cultured in control culture medium (CM) or
differentiation medium (DM) with or without TNF- for 7 days were determined and
mineralization ratio per cell was calculated. Data are shown as mean ± standard
deviation (n = 3). **p < 0.01.
Fig. 5. Contribution of TNFR1 to odontoblastic differentiation of sHDPCs treated with
TNF-. (A) Quantitative RT-PCR of TNFR1 in yHDPCs and sHDPCs. (B) sHDPCs were
transduced with TNFR1 siRNA (siTNFR1) or control siRNA (Cont), and suppression of
TNFR1 was confirmed by quantitative RT-PCR assays. (C) Alizarin red S staining of the
transduced sHDPCs cultured in control culture medium (CM) or differentiation medium
(DM) with or without TNF- (10 ng/ml) for 9 days. (D) The graph shows the
quantification of the Alizarin red S positive areas. (E) Quantitative RT-PCR of the
expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN in the transduced
sHDPC-3R in CM or DM with or without TNF- (10 ng/ml) for 5 days. Data are shown
as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Fig. 6. Contribution of TNFR1 to odontoblastic differentiation of yHDPCs treated with
TNF-. (A) yHDPCs were transduced with TNFR1 siRNA (siTNFR1) or control siRNA
(Cont), and suppression of TNFR1 was confirmed by quantitative RT-PCR assays. (B)
Alizarin red S staining of the transduced yHDPCs cultured in control culture medium
(CM) or differentiation medium (DM) with or without TNF- (10 ng/ml) for 9 days. (C)
The graph shows the quantification of the Alizarin red S positive areas. (D) Quantitative
RT-PCR of the expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN in
the transduced yHDPC-3R in CM or DM with or without TNF- (10 ng/ml) for 5 days.
Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
TABLE: Specific primer sequences, annealing temperature, cycle numbers, product sizes,
and sequence IDs for quantitative RT-PCR
Target gene
(abbreviation)
Forward (top) and reverse (bottom)
primer sequences
Size of
amplified
products
(bp)
Annealing
temperature(℃) Cycles Sequence ID
P16 CAACGCACCGAATAGTTACG
ACCAGCGTGTCCAGGAAG 171 60 40 XM_011517676.2
P21 GTCTTGTACCCTTGTGCCTC
GGCGTTTGGAGTGGTAGAAA 122 60 40 NM_001291549.1
P53 AGGAAATTTGCGTGTGGAGT
AGTGGATGGTTGTACAGTCA 107 60 40 NM_001126118.1
BSP ACTGGTGCCGTTTATGCCTTG
CTGGCACAGGGTATACAGGGTTAG 182 60 40 NM_004967.3
DSPP ATATTGAGGGCTGGAATGGGGA
TTTGTGGCTCCAGCATTGTCA 136 60 40 NM_014208.3
OCN CCCAGGCGCTACCTGTATCAA
GGTCAGCCAACTCGTCACAGTC 112 60 40 NM_199173.5
Nestin TGGCCACGTACAGGACCCTCC
AGATCCAAGACGCCGGCCCT 143 60 40 NM_006617.1
TNFR1 ATGCCGGTACTGGTTCTTCC
TGCCGAAAGGAAATGGGTCA 90 60 40 NM_001346091.1
TNF- CCTGCTGCACTTTGGAGTGA
GAGGGTTTGCTACAACATGGG 143 60 40 NM_000594.3
-actin ATTGCCGACAGGATGCAGA
GAGTACTTGCGCTCAGGAGGA 89 60 40 NM_001101.3
Senescence and odontoblastic differentiation of dental pulp cells
Supplementary Fig.1-7
Supplementary Figure 1. Senescence of cultured human dental pulp cells (HDPCs). (A,
D) HDPC-3Q (A) and HDPC-3S cells (D) were cultured until reaching the plateau in
proliferation and their population doublings (PDs) were determined. (B, E) SA--gal
activity in HDPC-3Q (B) and HDPC-3S cells (E) was examined. Only senescent cells were
observed to be blue. Bars: 100 µm. (C, F) The expression of aging-related genes p16, p21,
and p53 in HDPC-3Q (C) and HDPC-3S cells (F) was examined by quantitative RT-PCR
assays. Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Supplementary Figure 2. Comparison of the expression of odontoblast-related genes in
young human dental pulp cells (yHDPCs) and senescent HDPCs (sHDPCs). (A, B)
Quantitative RT-PCR for the expression of odontoblast-related genes BSP, DSPP, Nestin,
and OCN in HDPC-3Q (A) and HDPC-3S cells (B). Data are shown as mean ± standard
deviation (n = 3). **p < 0.01.
Supplementary Figure 3. Effects of TNF- on odontoblastic differentiation of human
dental pulp cells (HDPCs). (A, D, G, J) Alizarin red S staining of yHDPC-3Q (A), sHDPC-
3Q (D), yHDPC-3S (G) and sHDPC-3S cells (J) cultured in control culture medium (CM)
or differentiation medium (DM) with or without TNF- (10 ng/ml) for 7 days. (B, E, H,
K) The graphs show the quantification of the Alizarin red-S positive areas in yHDPC-3Q
(B), sHDPC-3Q (E), yHDPC-3S (H) and sHDPC-3S cells (K). (C, F, I, L) Quantitative RT-
PCR for the expression of odontoblast-related genes BSP, DSPP, Nestin, and OCN in
yHDPC-3Q (C), sHDPC-3Q (F), yHDPC-3S (I) and sHDPC-3S cells (L) cultured in CM or
DM with or without TNF- (10 ng/ml) for 5 days. Data are shown as mean ± standard
deviation (n = 3). *p < 0.05, **p < 0.01. sHDPC = senescent HDPC; yHDPC = young
human dental pulp cell.
Supplementary Figure 4. Comparison of mineralization ratio between young human
dental pulp cells (yHDPCs) and senescent HDPCs (sHDPCs). (A, B) Alizarin red S
positive areas in yHDPC-3Q (A), sHDPC-3Q (A), yHDPC-3S (B) and sHDPC-3S (B) cells
cultured in control culture medium (CM) or differentiation medium (DM) with or without
TNF- for 7 days were determined and the mineralization ratio per cell was then
calculated. Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01.
Supplementary Figure 5. Contribution of TNFR1 to odontoblastic differentiation of
sHDPCs treated with TNF-. (A, F) Quantitative RT-PCR for the gene expression of
TNFR1 in yHDPC-3Q (A), sHDPC-3Q (A), yHDPC-3S (F) and sHDPC-3S cells (F). (B, G)
sHDPC-3Q (B) and sHDPC-3S (G) cells were transduced with TNFR1 siRNA (siTNFR1)
or control siRNA (Cont), and suppression of TNFR1 was confirmed by quantitative RT-
PCR assays. (C, H) Alizarin red S staining of the transduced sHDPC-3Q (C) and sHDPC-
3S (H) cells cultured in control culture medium (CM) or differentiation medium (DM)
with or without TNF- (10 ng/ml) for 9 days. (D, I) The graph shows the quantification
of the Alizarin red S positive areas. (E, J) Quantitative RT-PCR for the expression of
odontoblast-related genes BSP, DSPP, Nestin, and OCN in the transduced sHDPC-3Q
(E) and sHDPC-3S cells (J) in CM or DM with or without TNF- (10 ng/ml) for 5 days.
Data are shown as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01. sHDPC =
senescent HDPC; yHDPC = young human dental pulp cell.
Supplementary Figure 6. Contribution of TNFR1 to odontoblastic differentiation of
yHDPCs treated with TNF-. (A, E) yHDPC-3Q (A) and sHDPC-3S (E) cells were
transduced with TNFR1 siRNA (siTNFR1) or control siRNA (Cont), and suppression of
TNFR1 was confirmed by quantitative RT-PCR assays. (B, F) Alizarin red S staining of
the transduced yHDPC-3Q (B) and yHDPC-3S (F) cells cultured in control culture
medium (CM) or differentiation medium (DM) with or without TNF- (10 ng/ml) for 9
days. (C, G) The graph shows the quantification of the Alizarin red S positive areas. (D,
H) Quantitative RT-PCR for the expression of odontoblast-related genes BSP, DSPP,
Nestin, and OCN in the transduced yHDPC-3Q (D) and yHDPC-3S cells (H) in CM or
DM with or without TNF- (10 ng/ml) for 5 days. Data are shown as mean ± standard
deviation (n = 3). *p < 0.05, **p < 0.01. sHDPC = senescent HDPC; yHDPC = young
human dental pulp cell.
Supplementary Figure 7. Comparison of the expression of TNF- in young human dental
pulp cells (yHDPCs) and senescent HDPCs (sHDPCs). Quantitative RT-PCR for the
expression of TNF- in HDPC-3R, HDPC-3Q and HDPC-3S cells. Data are shown as
mean ± standard deviation (n = 3). **p < 0.01.